It is estimated that one third of all vertebrate proteins are membrane bound. Unfortunately, by virtue of their hydrophobic nature, membrane proteins are often highly insoluble and are notoriously difficult to purify and crystallize. If the "Structural Genomics Initiative" is to succeed on a reasonable time scale, the X-ray structure determination of membrane proteins must be greatly simplified. Therefore, our goal is to develop an integrated set of research tools that are designed to enable the rapid cloning, expression, purification, crystallization and X-ray structure determination of membrane proteins in a generally applicable manner. Specifically, we propose to demonstrate that human topoisomerase I (topo I) can be used in to rapidly sub-clone PCR products into any commercial expression vector, in an orientation of choice. We also intend to demonstrate that a variety of membrane proteins can be made as chimeric proteins that are simple to express, purify, and co-crystallize with quasi-solid cationic lipid bilayers. Success in these endeavors will facilitate the high-throughput X-ray structure determination of not just membrane proteins, but any protein that may resist crystallization due to its hydrophobic nature. PROPOSED COMMERCIAL APPLICATION: If successful, this Phase I project would form the basis for a general and integrated approach towards the X-ray structure determination of membrane proteins. Moreover, since a major portion of industrial biotechnology is concerned with signaling pathways involving membrane receptor proteins, success in our approach would create a very large demand for the crystallography research services offered by Emerald BioStructures. Hence, the proposed research will not only facilitate the high-throughput structure determination of membrane proteins, but will help to spawn a new service industry in macromolecular crystallography.